Compounds and methods for reducing the recruitment and/or migration of polymorphonuclear cells

ABSTRACT

A therapeutically effective amount of an oligonucleotide is capable of influencing the properties and behavior of polymorphonuclear cells, e.g. suppressing endothelial adhesion and transmigration of said cells, and through this mechanism reduce the recruitment and/or migration of polymorphonuclear cells to a site of inflammation.

RELATED APPLICATIONS

The present application is a 371 of PCT/SE2009/051227 filed Oct. 28,2009 and claims priority under 35 U.S.C. 119 of U.S. Application No.61/111,284 filed Nov. 4, 2008.

TECHNICAL FIELD

The present inventions concerns methods and compounds for reducing therecruitment and/or migration of polymorphonuclear cells, and inparticular compounds and methods for the prevention, treatment oralleviation of various conditions where the properties and behaviour ofpolymorphonuclear cells play a role in the pathogenesis.

BACKGROUND

Inflammation is a general term for the mechanisms by which the bodyreacts to infection, irritation and other injury, mobilizing componentsof the immune system. Polymorphonuclear cells are recruited in the earlystages of inflammation, and migrate to the site of inflammation.Chemokines and their receptors, together with other chemoattractants,are key mediators for PMN migration. Examples of chemoattractants areIL-8 and LTB₄, which by binding to their receptors CXCR1/CXCR2 and BLT1,respectively, play a crucial role in the recruitment of PMN to the siteof inflammation. Importantly, inflammation plays a role in numerousconditions, not only diseases normally classified as inflammatorydiseases.

Ischemia is an absolute or relative shortage of the blood supply to anorgan that results in tissue damage because of a lack of oxygen andnutrients. The heart, the brain, and the kidneys are among the organsthat are the most sensitive to inadequate blood supply. Differenttreatment strategies are used depend on the organs involved and thecause of ischemia. One example is, after an acute heart ischemia(myocardial infarction), either a thrombolytic therapy or primarypercutaneous coronary intervention has to be used to restore blood flow(myocardial reperfusion) in the infarct-related coronary artery.However, the restoration of blood after an ischemic episode causes thedeath of cardiac myocytes that were viable immediately before myocardialreperfusion. This myocardial injury is termed lethal reperfusion injurywhich increases the final size of myocardial infarct. Myocardialischemia and reperfusion injury are believed to be associated withinflammatory reactions involving various types of cells and cytokines(Entman and Smith 1994).

Another example is stroke, and in particular ischemic stroke. In anischemic stroke, blood supply to one or more parts of the brain isdecreased, leading to dysfunction and necrosis of the brain tissue inthose parts. There are several underlying causes for an ischemic stroke:thrombosis (obstruction of a blood vessel by a blood clot forminglocally), embolism (obstruction of a blood vessel due to an embolusformed elsewhere in the body), systemic hypoperfusion (general decreasein blood supply, e.g. as a consequence of shock) and venous thrombosis.

Embolism is a serious condition which can lead to limited blood supplyto organs or tissues, downstream from the embolus. Embolism, mentionedabove as one causative factor in stroke, is known to cause obstructionin other organs, frequently in the lungs, kidneys, or liver, but also inthe lower limbs. An embolus can form spontaneously, for example whenplack is dislocated from the walls of a blood vessel and travels in theblood stream. Emboli may also form as a result of trauma, for examplefat emboli from complicated fractures or blood clots (thrombi) from thesite of haemorrhage. Patients undergoing surgery are also at risk, asboth thrombi and fat emboli may form during the surgical intervention.Also immobility, obesity and cancer are risk factors, known to beassociated with embolism.

Mesenteric ischemia is a medical condition in which inflammation andinjury of the small intestine result from inadequate blood supply.Causes of the reduced blood flow can include changes in the systemiccirculation (e.g. low blood pressure) or local factors such asconstriction of blood vessels or a blood clot. Other intestinaldisorders and conditions potentially leading to ischemia include ileus,distention, invagination, and volvolus, where abnormal orientation ofthe intestines, disruption of the peristaltic movement, and otherconditions can lead to reduced blood flow, inflammation, and eventuallyischemia. For example ileus may increase adhesion formation, becauseintestinal segments are in prolonged contact, allowing fibrous adhesionsto form, and intestinal distention can cause serosal injury andischemia. Such disorders can arise as a result of surgical intervention,either during the surgery or during recovery, as a result of trauma,burns, shock or various etiology etc and may lead to multiple organfailure.

Polymorphonuclear cells (PMNs), in particular polymorphonuclearneutrophils, which constitute the majority of the blood leukocytes aredrawn into the infarct zone by chemoattractants during the first 6 hoursof myocardial reperfusion, and during the next 24 hours they migrateinto the myocardial tissue. This process is facilitated by cell adhesionmolecules. The neutrophils cause vascular plugging and releasedegradative enzymes and reactive oxygen species (Vinten-Johansen J,2004). Therefore neutrophils are the primary target for the purpose ofthe treatment or prevention of inflammation. Several interventions wereaimed at reducing neutrophils from the infarct area during myocardialreperfusion e.g. leukocyte-depleted blood, antibodies against the celladhesion molecules, and pharmacologic inhibitors of complementactivation. However, the corresponding clinical studies have not shownany meaningful cardioprotective effect of such interventions (Reviewedin Yellon, 2007).

PMN accumulation and activation has been shown to play a central role inthe pathogenesis of a wide range of disease states as diverse asrheumatoid arthritis, atherosclerosis, ulcerative colitis, psoriasis,and ischemic damage. Hence the elucidation of endogenous regulatorymechanisms that can control neutrophil functions are of considerabletherapeutic interest. Extensive efforts have been spent on identifyingdrug candidates, and one approach is represented by the use of peptidecompounds, which bind to the αM integrin I-domain and inhibit itscomplex formation with proMMP-9, thereby preventing neutrophil migration(See e.g. WO2004/110477)

Another approach is the use of lipoxin and lipoxin derivatives, smalllipophilic compounds which have been shown to inhibit leukocyterecruitment and PMN infiltration in animal models of inflammation (Seee.g. WO2000/055109).

Yet another approach is the use of antibodies. In the early 1990-ties, apotent CD47-specific antibody (Ab), C5/D5, was identified that wascapable of inhibiting PMN migration across vascular endothelium,collagen-coated filters and intestinal epithelium without inhibiting β2integrin-mediated adhesion (Parkos, et al., 1996). At the same time, itwas shown that anti-CD47 also inhibited PMN migration across endothelialmonolayers (Cooper, et al., Proc Natl Acad Sci USA, 92: 3978, 1995).Subsequent studies with CD47 knockout mice have confirmed the importanceof CD47 in PMN migration in vivo suggesting that CD47 plays a role inregulating the rate of PMN recruitment to sites of infection. (Lindberget al., 1996).

Transplantation is another application where the consequences ofreperfusion ischemia must be considered. Transplantation means thetransfer of cells, tissue or parts of organs or entire organs from onelocation to another. Transplants can be autologous, so calledautografts, where mainly cells are taken from an individual and givenback to that same individual. More frequently, the term transplant isused for cells, tissue or organs taken from one person, the donor, andgiven to another person, the recipient. Kidney transplants are the mostcommonly performed. Transplants of the heart, liver and lungs are alsoregularly carried out. As medicine advances, other vital organsincluding the pancreas and small bowel are also being used intransplants. Tissue such as corneas, heart valves, skin and bone canalso be donated.

For practical reasons, a transplant needs to be stored outside the bodyfor a period of time, to allow for transport, functional testing, tissuetyping and matching the donor and the recipient. Since the advent oftransplantation, organs to be transplanted have been kept in coldischemic storage. Although this method was intended to help reduce theextent of organ damage during transport, significant damage stilloccurs. The more time that passes, the more serious damage. Differenttechnical and chemical solutions have been proposed. However, as thenumber of persons in need of a transplant far exceeds the number ofdonors, and as the procedure is very complicated, costly and stressfulfor all parties, there remains a need for improvements that increase thechance of a successful transplantation. Minimizing organ damage duringstorage and transport is an important issue.

WO 2005/080568 concerns the use of NF-kB inhibiting compounds for theprevention or reduction of the extent of secondary ischemic damage in amammal. The NF-kB inhibiting compounds are chosen from the groupconsisting of: an antisense NF-KB p65 subunit oligonucleotide; adominant-negative form of the NF-KB p65 subunit; a decoy; ribosomeinhibitors; enzymatic RNA against NF-KB p65; and siRNA constructs.

WO 2007/030580 concerns methods of protecting cells against cytotoxicinsult, involving the administration of a composition including an agentthat binds to and activates a Toll-like receptor to a subject,optionally in combination with administering an ASIC inhibitor. Themethods are stated to be applicable to the protection of neural andnon-neural cells. For example, methods of protecting a neural cellagainst excitotoxic brain injury are provided. Methods for preparingmedicaments for the prophylactic treatment of excitotoxic injury,ischemia and/or hypoxia are also provided.

WO 2007/030581 is a parallel application to the above WO 2007/030580,focussing on the administration of a CpG oligonucleotide for protectingcells against cytotoxic insult.

SUMMARY

The present inventors have surprisingly shown that specificoligonucleotide compounds influence the properties and behaviour ofpolymorphonuclear cells, in particular the recruitment and/or migrationof polymorphonuclear cells to a site of inflammation, and that theythrough this mechanism have utility in the prevention, treatment and/oralleviation of various diseases.

The inventors make available oligonucleotides and methods of their usein therapy, as well as in the manufacture of pharmaceutical compositionsfor this purpose. Clinical situations where it is desirable to preventor reduce the recruitment and/or migration of polymorphonuclear cells toa site of inflammation include, but are not limited to airwayinflammation, pleurisy, myocardial infarct, cerebral infarct, stroke,reperfusion injury related to tissue or organ transplants; andreperfusion injury related to surgical intervention, embolism, woundhealing, and trauma.

One aspect of the invention is the provision of novel compounds, as wellas methods of their use, such as methods for preventing or reducing therecruitment and/or migration of polymorphonuclear cells to a site ofinflammation in diseases of different aetiology. Other aspects of theinvention, together with their advantages, will be obvious to a skilledperson upon study of the claims, hereby incorporated by reference.

SHORT DESCRIPTION OF THE DRAWINGS

The invention will be disclosed in closer detail below, in thedescription, non-limiting examples, and claims, with reference to theenclosed drawings in which:

FIG. 1 a is the prophylactic protocol in the murine model of OVA inducedairway inflammation. The mice were sensitized with two i.p. injectionsof OVA in aluminium hydroxide gel on day 0 and 12. Treatments wereperformed with two intra nasal administrations of test drug or vehicleon day 16 and 21. The mice were then exposed to four aerosol challenges.The experiment was terminated on day 35.

FIG. 1 b is a histogram showing mean values of BAL fluid derived cellsafter treatment according to FIG. 1 a. Treatment with IDX9059 resultedin reduction of the leukocytes, mainly eosinophils, and lymphocytes. Barindicates mean±SD. *P<0.05, ***P<0.001 by One-way ANOVA with Bonferronipost hoc correction versus PBS control.

FIG. 2 shows the reduction of PMN accumulation after treatment withinventive compounds in a thioglycolate induced pleurisy model in mice. 2a) IDX9010 was administered 20 minutes before induction of pleurisy andresulted in a reduction of immigrating PMN's into the pleural cavitywith 68.2%. 2 b) IDX9059 was administered 20 minutes before induction ofpleurisy and resulted in a reduction of immigrating PMN's into thepleural cavity with 25.1%. Bars indicate mean±SD.

FIG. 3 a is a histogram of cells per microscope field withoutstimulation with any chemotactic factor in a model of intravitalmicroscopy of venules in the cremaster muscle in mice. R=rolling cells,A=adhering cells and T=transmigrated cells. n=4, mean±SD. Theunstimulated cells revealed a cell activity order of R>A>T. Treatmentwith IDX9059 showed down regulating effects on rolling and adherence ofunstimulated PMN cells.

FIG. 3 b is a histogram of cells per microscope field after addition ofchemotactic platelet activating factor (PAF) in a model of intravitalmicroscopy of venules in the cremaster muscle in mice. R=rolling cells,A=adhering cells and T=transmigrated cells. On stimulated cells, (afterPAF) the activity order was reversed, T>A>R>. Treatment with IDX9059showed down regulating effects on rolling and adherence of andtransmigration of PMN cells. n=4, mean±SD.

FIGS. 4 a-c show the effect of treatment with inventive drugs andvehicle (PBS) after induction of cerebral ischemia in rat model of focalischemia.

4 a) consists of two photographs showing a comparison of the extent ofischemic damage in non-treated animals given PBS (slide E) versusIDX9059 (slide D). The transient occlusion of middle cerebral artery wasused to induce ischemic brain damage in male Wistar Hannover rats. LaserDoppler was used to show the cortical blood flow of middle cerebralartery (MCA). After 90 minutes of occlusion the filament was removed andthe circulation of blood continued in the MCA and the cortical bloodflow restituted. The substances were injected intraperitoneal at 0 and24 hours after recirculation. The animals were sacrificed after 48 hoursof operation and the brain removed and cut into 2 mm thick slices. Theslices were then incubated in 0.8% triphenyltetrazolium chloride (TTC)in phosphate buffer to distinguish the viable brain cells (red) fromnecrotic (pale). The slices were photograph and the brain damageevaluated by computational analysis.

4 b) is a bar diagram showing the percentage of total brain damage.

4 c) is a bar diagram showing the percentage of selective nerve necrosis(SNN) area. Selective nerve necrosis is depicted as an estimation of thepart that has a slightly pink tone and considered to be the penumbraregion.

4 d) is a bar diagram showing the percentage of infarct region (ischemiccore). Data shown as mean±SD (n=4-8). Mean indicated with bar. *P<0.05obtained by unpaired t-test in treated group versus PBS control.

FIG. 5 is a bar diagram showing the percentage of infarct in an animalmodel of heart ischemia. IDX9059 (1 μg/μl) or placebo was given (100 μl)subcutaneously 24 hrs before excision of the heart. The hearts wereharvested and perfused for 20 minutes for stabilization. Global ischemiawas induced by stopping perfusion, followed by 120 minutes ofreperfusion. At the end of reperfusion the heart was removed and theleft ventricle was cut into four slices, each of one mm thick and wereincubated in 1% triphenyltetrazolium chloride in phosphate buffer todistinguish the viable cardiomyocytes. Photographs were taken from theslices and the infarct volume evaluated by computational analysis. Theresults are shown in a bar diagram showing the percentage of infarctdamage. The data show mean (n=8) and SEM. *P<0.05 was calculated byMann-Whitney test.

FIGS. 6 a-c show result of treatment with IDX0150 and vehicle (PBS) in amouse model of intestinal ischemia-reperfusion. The ischemia was inducedby 15 minutes ligation of mesentric artery followed by 3 hours ofreperfusion. The mouse received PBS (6 a), or IDX0150 (6 b) 20 minutesbefore induction of ischemia. 6 a-b) illustrate part of mouse digestivetract (stomach, left; small intestine; caecum and colon) afteradministration of PBS or IDX0150, respectively. The inflammatory scoreof mice treated with the IDX0150 (score 2) versus mice receiving PBS(score 6.5) is shown in 6 c.

FIGS. 6 d-g show the levels of myeloperoxidase (MPO) in intestinal andlung tissue in the mouse model of intestinal ischemia and reperfusioninjury. IDX0150 or PBS was administered subcutaneously 20 min beforeinduction of ischemia (6 d, and 6 f) or immediately after start ofreperfusion (6 e, and 6 g). After 3 h of reperfusion, small intestine (6d, and 6 e) and lungs (6 f, and 6 g) were homogenized and the levels ofMPO were analyzed with ELISA. Results are presented as mean values withstandard deviations.

FIGS. 7 a-f show the results of CXCR1 and CXCR2 expression on humanperipheral PMN after stimulation with different test substances. HumanPMN from 5 healthy blood donors were stimulated with 25 μM of testcompounds or with medium alone (untreated) for 3 h. Cells weresubsequently harvested and analyzed for CXCR1 and CXCR2 expression byflow cytometry. The fold changes in mean fluorescence intensity (MFI)for CXCR1 (7 a) and CXCR2 (7 c) or the fold changes in the % of CXCR1+(7 b) and CXCR2+ (7 d) CD66b+ PMN were calculated by normalizing the MFIor % positive PMN of corresponding untreated cells to 1 (dotted blackline). 7 e) illustrates the relative MFI of CXCR1+ PMN after stimulationwith 0.5, 10 and 25 μM of IDX9052, IDX9054 and IDX9059 (n=5). 7 f) showsthe relative MFI of CXCR1+ PMN after stimulation with 10 μM IDX9074 in aseparate experiments using PMN from 5 healthy blood donors. Results arepresented as mean±SEM. *P<0.05, **P<0.01 and ***P<0.001 were calculatedby two-way ANOVA with Bonferroni's post hoc correction versus untreatedcells.

FIG. 7 g illustrates CXCR1 expression on human PMN stimulated withIDX9059 for various time points. Human PMN from 3 healthy blood donorwere stimulated with 10 μM of IDX9059 for 15 min, 30 min, 1 h, 2 h or 3h. Cells were subsequently harvested and fixated at each time point andanalyzed for CXCR1 expression by flow cytometry. The fold changes in MFIof CXCR1 of CD66b positive PMN in IDX9059 treated cells were calculatedby normalizing the MFI of corresponding untreated cells to 1. Resultsare presented as mean values±SEM.

FIG. 7 h shows the importance of CpG and oligo G-oligonucleotides on thesurface expression of CXCR1. Human PMN from 3 healthy blood donors wereincubated for 3 h with 0.5, 10 or 25 μM of IDX9022 and IDX9059 as wellas the modified control oligonucleotides IDX0480 and IDX9134, which havethe same sequences as IDX9022 and IDX9059, respectively, but without CpGmotifs. Cells were subsequently harvested and analyzed for CXCR1expression by flow cytometry. The fold changes in MFI of CXCR1+ CD66b+PMN were calculated by normalizing the MFI of untreated cells to 1(dotted black line). Results are presented as mean±SEM.

FIG. 7 i demonstrates the effect of chloroquine on down-regulation ofCXCR1 surface expression after treatment with inventive compounds. HumanPMN from 4 healthy blood donors were pre-incubated for 30 min with 0.5,5 or 10 μg/ml of chloroquine before stimulated with 10 μM of testcompounds for 3 h. Cells were subsequently harvested and analyzed forCXCR1 expression by flow cytometry. The fold changes in MFI werecalculated by normalizing the MFI of corresponding untreated cells to 1(dotted black line). Results are presented as mean±SEM. *P<0.05 wascalculated by two-way ANOVA with Bonferroni's post hoc correction versusuntreated cells.

FIGS. 7 j-k show the results of BLT1 surface expression on human PMNafter stimulation with different test substances. Human PMN from 5healthy blood donors were stimulated with 0.5, 10 or 25 μM of inventivecompounds or with medium alone (untreated) for 3 h. Cells weresubsequently harvested and analyzed for BLT1 expression by flowcytometry. The fold change in MFI (7 j) or % BLT1+ CD66b positive PMN (7k) were calculated by normalizing the MFI or % of correspondinguntreated cells to 1 (dotted black line). Results are presented as meanand SEM. *P<0.05, **P<0.01 and ***P<0.001 were calculated by two-wayANOVA with Bonferroni's post hoc correction versus untreated cells.

FIG. 7 l-n show the effect of the inventive compounds on IL-8 and LTB₄induced chemotaxis of PMN. Human PMN from 5 (7 l) or 4-6 (7 n) healthyblood donors were preincubated with 0.5, 10 or 25 μM of inventivecompounds for 1 h after which free compound were washed away and thecells were investigated for their ability to migrate towards IL-8 (7 l)or LTB₄ (7 n) in a chemotaxis assay for 3 h. Results are presented asthe mean number of migrated PMN±SEM. *P<0.05 and **P<0.01 werecalculated by one-way ANOVA with Dunnett's post hoc correction versusuntreated cells incubated with IL-8/LTB₄. 7 m) shows a separateexperiment where PMN from 2 healthy blood donors were preincubated with0.5, 10 or 25 μM of IDX9045 for 1 h, after which the cells wereinvestigated for their ability to migrate towards IL-8 in the presenceof free compound in a chemotaxis assay for 3 h.

FIGS. 7 o-q demonstrate the correlation between CXCR1, CXCR2 and BLT1surface expression and PMN migration after stimulation with theinventive compounds. The MFI of CXCR1 (7 o), CXCR2 (7 p) and BLT1 (7 q)of PMN after stimulation with inventive compounds were plotted againstthe number of PMN that migrated towards IL-8 (7 o and 7 p) or LTB₄ (7 q)in the chemotaxis assay. The curve fit (r²) is specified in the figures.

FIGS. 8 a-f show the results of CXCR1, CXCR2 and BLT1 expression onhuman peripheral PMN isolated from MS patients after stimulation withdifferent test substances. Human PMN from MS patients were stimulatedwith 0.5, 10 and 25 μM of test compounds or with medium alone(untreated) for 3 h. Cells were subsequently harvested and analyzed forCXCR1 (n=4), CXCR2 (n=4) and BLT1 (n=2) expression by flow cytometry.The fold changes in mean fluorescence intensity (MFI) for CXCR1 (8 a),CXCR2 (8 c) and BLT1 (8 e) or the fold changes in the % of CXCR1+ (8 b),CXCR2+ (8 d) and BLT1+ (8 f) CD66b+ PMN were calculated by normalizingthe MFI or % positive PMN of corresponding untreated cells to 1 (dottedblack line). Results are presented as mean±SEM. *P<0.05, **P<0.01 and***P<0.001 were calculated by two-way ANOVA with Bonferroni post hoccorrection versus untreated cells.

FIGS. 8 g-i illustrate the results of CXCR1, CXCR2 and BLT1 expressionon human peripheral PMN derived from an asthmatic patient afterstimulation with the inventive compounds. Human PMN from an asthmaticpatient were stimulated with 0.5, 10 and 25 μM of test compounds or withmedium alone (untreated) for 3 h. Cells were subsequently harvested andanalyzed for CXCR1, CXCR2 and BLT1 expression by flow cytometry. Thefold changes in mean fluorescence intensity (MFI) for CXCR1 (8 g), CXCR2(8 h) and BLT1 (8 i) CD66b+ PMN were calculated by normalizing the MFIof corresponding untreated cells to 1 (dotted black line).

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthe terminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

Also, the term “about” is used to indicate a deviation of +/−2% of thegiven value, preferably +/−5%, and most preferably +/−10% of the numericvalues, where applicable.

In addition to the above, the following terms will be used:

The term “homology” and “degree of homology” means the similarity oridentity between two sequences, where 100% homology means that thesequences are identical, and a lower homology indicates the presence ofvariations. For example, for a sequence consisting of 20 nucleobases,another sequence is 90% homologous if 18 bases are the same.

“Functionally homologous” means sequences sharing perhaps a lowerstructural homology with the disclosed sequence, but exhibitinghomologous function in vivo, in either the healthy or the diseasedorganism, e.g. coding the same or highly similar proteins with similarcellular functions.

The terms “treatment”, “therapy”, “therapeutic use”, “medicament”, and“medical use” encompass both human and animal or veterinaryapplications.

The present inventors have found that specific oligonucleotides arecapable of influencing the properties and behaviour of polymorphonuclearcells, e.g. their endothelial adhesion and transmigration, and thatthese compounds through this and possibly other mechanisms have utilityin the prevention and/or alleviation of diseases of different etiology.These findings have been confirmed in animal experiments and in in vitrotests performed on PMNs isolated from donor blood.

TABLE 1 Examples of olignucleotide sequences SEQ ID NO IDX-No Seq 5′-3′ 1 IDX9005 T*C*G*TCCATGGTCA GGGTCCCGG*G*G*G  2 IDX9010T*C*C*CAAGAGTCGTCC*A*G*G  3 IDX9022 T*C*G*TCGTTCTGCCATCGTC*G*T*T  4IDX9030 T*C*G*TCTGCCATGGCGGCC*G*C*C  5 IDX9031T*C*G*TCGATTCGTCTGCCA*T*G*G  6 IDX9045 G*G*G*TCGCAGC*T*G*G  7 IDX9054G*G*G*GTCGTCTGC*G*G*G  8 IDX9059 G*A*T*CGTCCG*G*G*G  9 IDX9074T*C*G*TTCGTCTTTCGTC*T*G*C 10 IDX9092 T*T*T*CGTCTGCTTTCGTTTCG*T*T*T 11IDX9095 T*C*G*TCTGCTTTCGTC*T*G*C 12 IDX9096 G*A*T*CGTCCGATCG*T*C*C 13IDX0150 G*G*A*ACAGTTCGTCCAT*G*G*C 14 IDX9052 G*G*G*GTCGTCTG*C*G*G 15IDX0480 T*G*C*TGCTTCTGCCATGCTG*C*T*T 16 IDX9134 G*A*T*GCTCTG*G*G*G

The inventors make available novel oligonucleotides, and accordingly oneembodiment of the invention is an isolated and substantially purifiedoligonucleotide chosen from the group consisting of SEQ ID NO 1-12, 14,15, and 16, and in particular SEQ ID NO 8 [IDX9059]; SEQ ID NO 14[IDX9052]; SEQ ID NO 7 [IDX9054]; SEQ ID NO 6 [IDX9045]; SEQ ID NO 1[IDX9005]; SEQ ID NO 9 [IDX9074]; SEQ ID NO 3 [IDX9022]; SEQ ID NO 2[IDX9010]; and SEQ ID NO 4 [IDX9030].

Preferably at least one nucleotide in a sequence chosen from thesequences above has a phosphate backbone modification. Said phosphatebackbone modification is preferably a phosphorothioate orphosphorodithioate modification.

The inventors also make available a pharmaceutical compositioncomprising an oligonucleotide chosen from the sequences above.

One embodiment of the invention is the use of an oligonucleotide for theproduction of a pharmaceutical preparation for reducing the recruitmentand/or migration of polymorphonuclear cells to a site of inflammation,wherein the oligonucleotide is chosen from the group consisting of: SEQID NO 8 [IDX9059]; SEQ ID NO 14 [IDX9052]; SEQ ID NO 7 [IDX9054]; SEQ IDNO 6 [IDX9045]; SEQ ID NO 1 [IDX9005]; SEQ ID NO 9 [IDX9074]; SEQ ID NO3 [IDX9022]; SEQ ID NO 2 [IDX9010]; SEQ ID NO 4 [IDX9030]; and SEQ ID NO13 [IDX0150].

Preferably, according to the results currently available to theinventors, said reduced recruitment and/or migration ofpolymorphonuclear cells to a site of inflammation is a result of adown-regulation of at least one of the receptors CXCR1 and CXCR2, or aresult of a down-regulation of the receptor BLT1.

According to a preferred embodiment, said oligonucleotide is preferablychosen among: SEQ ID NO. 8 [IDX9059]; SEQ ID NO. 14 [IDX9052]; and SEQID NO. 7 [IDX9054].

In the above use, the oligonucleotide is preferably given through one ofthe following routes of administration: subcutaneous, intraperitoneal,mucosal, intestinal, oral, gastric, oesophagal, buccal, nasal, andpulmonary administration.

According to a preferred embodiment, freely combinable with the above,the oligonucleotide is administered as a step in the treatment of aninflammatory disease.

According to another preferred embodiment, freely combinable with theabove, the oligonucleotide is administered to a patient having sufferedfrom, or suspected to have suffered from, a myocardial infarct.

According to another preferred embodiment, freely combinable with theabove, the oligonucleotide is administered to a patient having sufferedfrom, or suspected to have suffered from, a stroke.

According to another preferred embodiment, freely combinable with theabove, the oligonucleotide is administered to a patient having sufferedfrom trauma or burn, or scheduled to undergo surgery.

According to another preferred embodiment, freely combinable with theabove, the oligonucleotide is administered to a patient having anembolism before removal of the embolism or before administration of athrombolytic agent. As the compounds have been shown to have also apost-conditioning effect, it is contemplated that they are administeredalso after the removal of the embolism or administration of athrombolytic agent, something that significantly increases theirusefulness in an intensive care setting.

According to another preferred embodiment, freely combinable with theabove, the oligonucleotide is administered to an organ designated fortransplantation, either in situ, before extraction from the donor, intransit before implantation in the recipient, or in vivo, before or atthe time of restoring the blood flow.

Further, the inventors make available a method for reducing therecruitment and/or migration of polymorphonuclear cells to a site ofinflammation in an organ of a human patient, wherein the oligonucleotidechosen from the group consisting of: SEQ ID NO 8 [IDX9059]; SEQ ID NO 14[IDX9052]; SEQ ID NO 7 [IDX9054]; SEQ ID NO 6 [IDX9045]; SEQ ID NO 1[IDX9005]; SEQ ID NO 9 [IDX9074]; SEQ ID NO 3 [IDX9022]; SEQ ID NO 2[IDX9010]; SEQ ID NO 4 [IDX9030]; and SEQ ID NO 13 [IDX0150], isadministered locally to said organ or systemically to said patient.

Alternatively, the inventors make available a method for reducing therecruitment and/or migration of polymorphonuclear cells to a site ofinflammation in an organ, wherein an oligonucleotide is administeredlocally to said organ or systemically to said patient before,simultaneously with, or after reperfusion of said organ, wherein theoligonucleotide is chosen from the group consisting of: SEQ ID NO 8[IDX9059]; SEQ ID NO 14 [IDX9052]; SEQ ID NO 7 [IDX9054]; SEQ ID NO 6[IDX9045]; SEQ ID NO 1 [IDX9005]; SEQ ID NO 9 [IDX9074]; SEQ ID NO 3[IDX9022]; SEQ ID NO 2 [IDX9010]; SEQ ID NO 4 [IDX9030]; and SEQ ID NO13 [IDX0150].

In either of the above methods of treatment, and according to theresults currently available to the inventors, said reduced recruitmentand/or migration of polymorphonuclear cells to a site of inflammation isa result of a down-regulation of at least one of the receptors CXCR1 andCXCR2, or a result of a down-regulation of the receptor BLT1.

In the above methods of treatment, said oligonucleotide is preferablychosen among: SEQ ID NO. 8 [IDX9059]; SEQ ID NO. 14 [IDX9052]; and SEQID NO. 7 [IDX9054].

The inventors also contemplate a method for conditioning a patienthaving suffered or suspected of having suffered from a disturbance orinterruption in the blood flow in an organ and scheduled for treatment,wherein an oligonucleotide capable of reducing the recruitment and/ormigration of polymorphonuclear cells to a site of inflammation in saidorgan, is administered to said patient before, simultaneously with, orafter the scheduled treatment, wherein the oligonucleotide is chosenfrom the group consisting of: SEQ ID NO 8 [IDX9059]; SEQ ID NO 14[IDX9052]; SEQ ID NO 7 [IDX9054]; SEQ ID NO 6 [IDX9045]; SEQ ID NO 1[IDX9005]; SEQ ID NO 9 [IDX9074]; SEQ ID NO 3 [IDX9022]; SEQ ID NO 2[IDX9010]; SEQ ID NO 4 [IDX9030]; and SEQ ID NO 13 [IDX0150].

In an embodiment of the above method, said organ is preferably theheart, and said reduction of the recruitment and/or migration ofpolymorphonuclear cells to a site of inflammation in said organ iseffective to reduce ischemic damage and prevent or alleviate secondaryreperfusion injury. In this embodiment, said secondary reperfusioninjury may be an injury following from restoring the blood flow to theheart through the administration of a thrombolytic agent. Alternatively,said secondary reperfusion injury is an injury following from restoringthe blood flow to the heart through surgical intervention, for exampleby-pass surgery. Alternatively, said secondary reperfusion injury is aninjury following from restoring the blood flow to the heart throughballoon angioplasty. Alternatively, said secondary reperfusion injury isan injury following from surgically restoring the blood flow to atransplanted organ in the recipient of the transplant.

In another embodiment of the above method, said organ is the brain, andsaid ischemic damage is a secondary reperfusion injury. In thisembodiment, said secondary reperfusion injury may be an injury followingthe restoration of the blood flow to the brain through theadministration of a thrombolytic agent.

In yet another embodiment of the above method, said organ is chosenamong the liver, at least one kidney, the intestines or parts thereof,at least one lung or parts thereof, and said ischemic damage is asecondary reperfusion injury.

In any of the above embodiments of the method, the oligonucleotide isgiven through one of the following routes of administration: systemic,preferably subcutaneous, intraperitoneal, mucosal, including intestinal,oral, gastric, oesophagal, buccal, nasal, and pulmonary administration.

The inventors also make available an adjuvant method for the treatmentof myocardial infarction, wherein an oligonucleotide chosen from thegroup consisting of: SEQ ID NO 8 [IDX9059]; SEQ ID NO 14 [IDX9052]; SEQID NO 7 [IDX9054]; SEQ ID NO 6 [IDX9045]; SEQ ID NO 1 [IDX9005]; SEQ IDNO 9 [IDX9074]; SEQ ID NO 3 [IDX9022]; SEQ ID NO 2 [IDX9010]; SEQ ID NO4 [IDX9030]; and SEQ ID NO 13 [IDX0150], is administered before, afteror simultaneously with the administration of a thrombolytic agent.

Further, the inventors make available an adjuvant method for thetreatment of stroke, wherein an oligonucleotide chosen from the groupconsisting of SEQ ID NO 8 [IDX9059]; SEQ ID NO 14 [IDX9052]; SEQ ID NO 7[IDX9054]; SEQ ID NO 6 [IDX9045]; SEQ ID NO 1 [IDX9005]; SEQ ID NO 9[IDX9074]; SEQ ID NO 3 [IDX9022]; SEQ ID NO 2 [IDX9010]; SEQ ID NO 4[IDX9030]; and SEQ ID NO 13 [IDX0150] is administered before, after orsimultaneously with the administration of a thrombolytic agent.

Another embodiment of the invention is a technical solution for thestorage and/or transport of transplants, wherein said solution comprisesan oligonucleotide capable of influencing the properties and behaviourof polymorphonuclear cells, e.g. suppressing endothelial adhesion andrecruitment and/or migration of polymorphonuclear cells, in an amountsufficient for the prevention and/or alleviation of ischemic damage, andsaid oligonucleotide is chosen among SEQ ID NO 8 [IDX9059]; SEQ ID NO 14[IDX9052]; SEQ ID NO 7 [IDX9054]; SEQ ID NO 6 [IDX9045]; SEQ ID NO 1[IDX9005]; SEQ ID NO 9 [IDX9074]; SEQ ID NO 3 [IDX9022]; SEQ ID NO 2[IDX9010]; SEQ ID NO 4 [IDX9030]; and SEQ ID NO 13 [IDX0150].

In the above methods of use, as well as the methods of treatment, theoligonucleotide is administered in a therapeutically effective dose. Thedefinition of a “therapeutically effective dose” is dependent on thedisease and treatment setting, a “therapeutically effective dose” beinga dose which alone or in combination with other treatments results in ameasurable improvement of the patient's condition. A skilled person candetermine a therapeutically effective dose either empirically, or basedon laboratory experiments, performed without undue burden. The treatingphysician can also determine a suitable dose, based on his/herexperience and considering the nature and severity of the disease, aswell as the patient's condition.

According to one embodiment, the oligonucleotide is given in a dose inthe interval of about 1 to about 2000 μg/kg bodyweight, preferably about5 to about 1000 μg/kg bodyweight, most preferably in a dose in theinterval of about 10 to about 500 μg/kg bodyweight.

The oligonucleotide may be administered in a single dose or in repeateddoses. The currently most preferred embodiment entails one single doseof the oligonucleotide according to the invention, administered to amucous membrane, e.g. given intranasally, orally, rectally orintravaginally in an amount of less than about 2000 μg, preferably lessthan about 500 μg, preferably about 100 μg per kg bodyweight.

Another currently preferred embodiment is the administration of theoligonucleotide in two or three doses, separated in time by about 2,about 6, about 12, or about 24 hours.

According to another embodiment of the invention, the oligonucleotide isadministered to an organ designated for transplantation, either in situ,before extraction from the donor, in transit before implantation in therecipient, or in vivo, before or at the time of restoring the bloodflow. Preferably the oligonucleotide is present in a concentration ofabout 0.1 to about 2000 μg/l in a solution used for conditioning thetransplant before extraction from the donor. Alternatively, or inaddition thereto, the oligonucleotide is present in a concentration of0.1 to about 1000 μg/l in a solution used for transporting thetransplant. Alternatively, or in addition thereto, the oligonucleotideis present in a concentration of 0.1 to about 1000 μg/l in a solutionused for conditioning the transplant before restoring blood flow.

There are indications that the oligonucleotide can be administered notonly before, but also simultaneously with, or even after restoration ofthe blood flow. In the context of Example 4, the possibility ofpre-conditioning, as well as post-conditioning is discussed. The resultssurprisingly show that treatment with the SEQ ID NO 8 [IDX9059] alsoafter induction of the stroke can serve as post-conditioning and protectthe brain against ischemic injury.

The above embodiments are mutually inclusive, meaning that differentdoses, modes of administration and time intervals may be freely combinedwithin the embodiments listed, as well as with other embodiments whichbecome apparent to a skilled person.

The above embodiments offer many advantages in that will be evident to askilled person upon study of the description and examples. One advantageis that the use of the oligonucleotides offers the possibility toreplace or to complement currently used drugs, and reduce adverseeffects associated with current drugs and treatments.

EXAMPLES 1. Nasal Administration of Immunomodulatory OligonucleotideTest Substance in a Murine Model of OVA Induced Airway Inflammation

Materials and Methods

Animals

Female Balb/c mice (8 weeks), obtained from B&K Sollentuna, Stockholm,Sweden, were used in the experiment. The mice were fed with a completepellet rodent diet, R36 (Laktamin AB, Stockholm, Sweden) and water ofdomestic drinking quality ad libitum. The animals were kept in animalrooms at 21° C., ±3° C., and with a relative humidity of 55%±15%. Theventilation system was designed to give 10 air changes per hour. Theroom was illuminated to give a cycle of 12 hours light and 12 hoursdarkness. Light is on from 07:00 h to 19:00 h. The mice were kept intransparent polycarbonate (Macrolone type III) cages (floor area: 820cm²), 5 in each cage. The bedding in the cages was 4HV Aspen bedding(Tapvei, Finland). Each cage was identified by a cage card marked withstudy number, group number, sex and animal numbers.

Sensitisation and Aerosol Challenge

Mice were sensitized intraperitoneally with 200 μL OVA/aluminiumhydroxide gel (1:3) on day 0 and 12 (FIG. 1 a). OVA (chicken egg albumingrade V, Sigma, St. Louis, Mo.) was dissolved in saline and mixed withaluminium hydroxide gel to a concentration of 50 μg/mL by rotation at 4°C. for 3 h. On days 23, 26, 30 and 33 (see FIG. 1 a), mice werechallenged in the lungs by inhalation of aerosolized OVA for 30 minutesusing a Batelle exposure chamber. Aerosols were generated by acompressed-air neubulizer (Collison 6-jet) at airflow 7.4 L/min using anebulizer concentration of 10 mg/mL OVA dissolved in PBS (Sigma, StLouis, Mo., USA). The control group with non-sensitized animals receivedno other treatment than aerosolized OVA at day 23, 26, 30 and 33. Therewas also a control group of sensitized mice which did not receiveaerosol challenge.

Oligonucleotide

In this OVA model (FIG. 1 a) a selected oligonucleotide, IDX9059, (SEQID NO 8, Table 1) was tested. The oligonucleotides were synthesized bybiomers.net GmbH, Ulm, Germany and stored frozen at −20° C.

Formulation

The immunomodulatory oligonucleotide was dissolved in phosphate bufferedsaline (PBS, Fluka Biochemika Ultra, Sigma Aldrich, St. Louis, USA).

Treatment of OVA Induced Airway Inflammation

In this experiment, intranasal instillation of IDX9059 (1,247 μg/μL) onday 16 and 21 in a prophylactic setting (FIG. 1 a) was given. Thesubstance was administered in 40 μL PBS giving a dose of ˜50 μg/mice(49.88 μg/mice). The two sensitized sham-treatment groups were instilledwith PBS, the same total volume as for the test compound on day 16 and21.

Analysis of Airway Inflammation Parameters

Mice were killed by cervical dislocation 42 h after the last OVA aerosolchallenge. The trachea was cannulated with polyethylene tubing (BectonDickinson, Sparks, Md., USA) and bronchoalveolar lavage (BAL) wasperformed using 4×1 mL aliquots of ice-cold Hank's balanced saltsolution (HBSS) (Sigma, St Louis, Mo., USA). The BAL fluid wascentrifuged (400 g, 10 min, 4° C.) and the supernatant recovered andfrozen for later analyses. The cells were resuspended in 0.4 mL PBS andthe total number of leukocytes counted using trypan blue exclusion in aBürker chamber. Duplicate Cytospin (Cytospin 3, Shandon, Runcorn, UK)preparations of BAL fluid cells were stained with May Grünewald Giemsastain for differential counts, using standard morphological criteria.

Statistical Analysis

Statistical comparisons were performed using One-way analysis ofvariance (ANOVA) using Dunnett's post hoc correction to compare withsensitized PBS treated control mice (GraphPad Prism 4). Data are shownas mean±standard deviation. A P-value below 0.05 was consideredsignificant.

Results

The ovalbumin induced allergic asthma model is a widely used model toreproduce the airway eosinophilia, pulmonary inflammation and elevatedIgE levels found during asthma. Analysis of this model relies on generalindicators of asthma such as BAL analysis where the type and amount ofinfiltrating inflammatory cells are identified and counted respectively.

Consequently, the BAL fluid cells derived from each mouse were countedas described and the values plotted as a combined histogram providingmean values for the different groups (FIG. 1 b).

In general terms, the level of induced airway inflammation was high asindicated by a large influx of the 4 analyzed cell types into the lungsof the animals (group PBS). The control groups demonstrated no signs ofinduced inflammation confirming that the animals did not exhibit anatural allergic response to the aerosol ovalbumin protein and that theovalbumin protein used was not contaminated with, for example, LPS.

The complete absence of any signs of inflammation in the “no aerosol”control groups confirmed that the experimental procedure of OVAimmunization itself does not induced lung inflammation.

Following nasal treatment the test substance SEQ ID NO 8 [IDX9059] had areducing effect on inflammatory cells, i.e. leukocytes (P<0.001),eosinophils (P<0.001) and lymphocytes (P<0.05).

In this in vivo model of allergic asthma leukocyte i.e. PMN mainlyeosinophil play an important role in lung inflammation. A statisticallysignificant reduction in the number of leukocytes, eosinophils andlymphocytes infiltrating the BAL fluid was observed in animals whentreated with the inventive compound SEQ ID NO 8 [IDX9059]. Thisindicates that the inventive compound is able to prevent inflammationthrough inhibition of PMN infiltration.

2. Thioglycolate Induced Pleurisy in C57/BI6 Mice

Materials and Methods

An animal model was set up to study the effect of oligonucleotidesaccording to an embodiment of the present invention on cell migrationand vascular permeability.

Mice were anesthetized by an intraperitoneal injection of 0.15-0.20 mlof a mixture of ketamine (Ketalar® Parke-Davis; 25 mg/ml) and xylazine(Narcoxyl Vet.® Veterinaria AG; 5 mg/ml).

The left jugular vein was cannulated with polyethylene tubing (PE10) forintravenous administration (i.v.). A skin incision was made on the rightside of the chest. Following dissection of the underlying muscle,pleurisy (inflammation of the lung sack) was induced by a singleintrapleural injection of 100 μl of thioglycolate (Sigma). Sterile PBSwas used as negative control. FITC-conjugated dextran in PBS (100 μl, 30mg/ml) was injected i.v. After 4 h, the animals were euthanized with anoverdose of anaesthesia, the chest was carefully opened and the exudatewas removed by aspiration and the volume noted. The thorax was thenrinsed with 1 ml of ice-cold 3 mM EDTA in PBS. Exudate which wascontaminated with red blood cells was discarded.

The exudate and rinse material was centrifuged at 1500 g for 5 min andthe supernatant was used for measurement of fluorescence intensity in afluorometer (Fluoroskan Acsent, LabSystems) and clearance volume ofFITC-dextran was calculated. The pellet was resuspended in PBS with 0.1%BSA for 15 min to block unspecific antibody binding. 10 μl of cellsuspension was used for differential WBC count in a Bürker chamber.

Cells from the exudate were stained with neutrophil and macrophagespecific antibodies and were analyzed by flow cytometry (FACSort andCellQuest software, BD). Analysis included total white blood cell count,based on their typical appearances in the forward and side scatter. PMNand macrophages were further identified by their expression of Ly6G andF4/80, respectively.

In order to test the anti inflammatory effect of oligonucleotides byreducing PMN migration according to embodiments of the presentinvention, the compounds to be tested were administeredintraperitoneally, at a dose of 100 μl, i.e 50 μg/mouse, about 20minutes before induction of pleurisy. In this study, IDX9010, IDX9054,IDX9059, (SEQ ID NO 2, 7, and 8, respectively, Table 1) and IDX 0150(SEQ ID NO 13) were tested.

Results

The thioglycolate induced pleurisy model is one of the models of choicefor practical screening of new drugs under development although it istechnically complicated and can show occasional individual disparatevalues. However, this model is restricted in the number of animals thatcan be tested simultaneously.

Results showed that animals responded to the inflammation inducingagent, thioglycolate, by a high immigration of PMN into the pleuralcavity, and the accumulation of pleural edema.

Mice (n=4) receiving IDX9010 showed a reduced number of PMN's by 68.2%(FIG. 2 a), but no reduction in edema (data not shown). Mice givenIDX9054 (n=4) showed no reduction in numbers of PMN, but could reducethe edema by 36% (data not shown). IDX9059 showed reduction in both PMNaccumulation (25.1%, FIG. 2 b), and edema (31%) (data not shown) in agroup of 7 animals. Mice (n=5) given IDX0150 showed 40.9% reduction inPMN-numbers, and 68.2% reduction in edema (data not shown). The resultsrepresent mean±SD.

Experiments using an anti-PMN antibody showed that the anti-PMN-ab couldequally reduce PMN and edema to the similar levels as the inventivecompounds (data not shown). The prophylactic administration of inventivecompounds in thioglycolate induced pleurisy led to reduction of PMNmigration into the pleural cavity.

3. Intravital Microscopy in a Murine Model of Vascular Inflammation

The in vivo, anti-inflammatory effect of oligonucleotide IDX9059 (SEQ IDNO 8, Table 1) on leukocyte extravasation in mice was investigated. Inresponse to a chemotactic stimulus, inflammatory cells transmigrate thewalls of blood vessels towards a gradient of stimulus. To transmigrate,the cells first must intermittently adhere to the vessel inner walls,i.e. rolling (R), thereafter start to adhere more firmly, adherence (A),and then migrate out in the surrounding tissue, transmigration (T).Platelet activation factor (PAF) was used to induce this inflammatoryprocess. In normal unstimulated vessels the order of these events wereR>A>T. After exposure to PAF this order was reversed, T>A>R, showingthat cells started to adhere and transmigrate.

Introduction

Leukocyte mobilisation is a prerequisite for an inflammatory response.In blood vessels a series of events between white blood cells andendothelium lead to accumulation of inflammatory cells at a site ofinjury or inflammation (Lindbom, 1983). One important cell in thiscascade of events is the polymorphonuclear cell (PMN). PMN's aretransported in the blood stream. The blood stream has a higher velocityin the center of the blood vessel and a lower speed towards the marginwhich allows for contact between the PMN and blood vessel wall.Molecular mechanisms facilitates adherence of PMN's to the endothelium(Penberthy et al., 1997). One group of such molecules are termedselectins. At first this interaction is only partial causing the PMN'sto roll along the endothelium. Stimulated by pro-inflammatory molecules,this adherence becomes more firm causing the PMN's to adhere to theendothelium, a phenomenon termed sticking. Sticking allows the PMN's toactively transmigrate the endothelial layer and subsequently enter theconnective tissue and are further directed by a gradient of inflammatoryfactors towards the epicenter of inflammation.

All these events can be studied in vivo by intravital microscopy.Intravital microcopy allows for the study of cells in small vessel, andin the surrounding connective tissue.

Suitable venules have been studied for example in the tenuissimus musclein rabbits, and in the cremaster muscle of mice.

This example deals with intravital microscopy in the cremaster muscle ofmice. The cremaster muscle is the muscle that controls the temperatureof the testis in the scrotum. The vasculature of this muscle can readilybe exposed under a water immersion objective, super fused with bufferedsaline at body temperature. Chemotactic or pro-inflammatory substancescan be added to the superfusing buffer and pharmacological can be addedto the superfusate or into the animal.

Rolling, sticking and transmigration can thereafter be studied by aid oftime-lapse video recording and recordings can later be used forquantification of cells or measurements of distances transversed.

The aim of this study was to study effects of the inventive compoundIDX9059 on inflammatory cell migration in venules after PAF stimulationin the mouse.

Materials and Methods

Animal material and conditions: C57BL/6 SPF mice from Scanbur AB,Sollentuna, Sweden, were kept in an animal room with controlledtemperature (21±2° C.), light-dark cycles of 12 h each, and were allowedfree access to food and water.

Test compound: The oligonucleotide DEQ ID NO 8 [IDX9059] was synthesizedby biomers.net GmbH, Ulm, Germany under non-GMP conditions and providedin PBS solution. The oligonucleotide was stored at −20° C. as stocksolutions and prepared 2-3 days prior to the initiation of theexperiment.

Formulation: The oligonucleotide was further diluted in PBS (Fluka,Sigma) at room temperature. The concentration was adjusted by aid of UVspectrophotometry (SmartSpec™ 3000, BIO-RAD, Hercules, USA) to 95%accuracy, until the desired concentration was reached.

Treatment of PAF induced inflammation: The animals were given IDX9059s.c., 50 μg/100 μl/mouse, circa 20 minutes before induction ofinflammation.

Intravital microscopy of leukocyte recruitment: Groups of 4 mice wereused. Mice were anesthetized by an intraperitoneal injection of0.15-0.20 ml of a mixture of ketamine (Ketalar®; Pfizer AB, Sollentuna,Sweden; 25 mg/ml) and xylazine (Narcoxyl Vet.®; Intervet InternationalB.V., Netherlands; 5 mg/ml). The left jugular vein was cannulated withpolyethylene tubing (PE10) for continuous administration of anesthesia.A ventral incision was made on the right scrotum and one testiswithdrawn. The cremaster muscle was dissected free of fascia, incised,and pinned out flat on a transparent pedestal to allowtrans-illumination. The testis was then pinned to the side. Thepreparation was kept moist and warm by continuous superfusion of a 37°C. temperature controlled bicarbonate buffer, maintaining physiologicallevels of temperature, pH, and gas tensions. Leukocyte extravasation wasinduced by addition of platelet activating factor (PAF, Sigma-Aldrich,St. Louis, Mo., USA; 100 nM) to the superfusion solution for 60 min.Measurements of rolling, adhesion and transmigration were made beforeand after stimulation. Video recordings were obtained from 20-50 μm widewell defined venules. Rolling was determined as the number of leukocytespassing a reference line perpendicular to blood flow during 30 seconds.Cells within the vessel were classified as adherent if they remainedstationary for more than 30 seconds. Transmigrated cells were counted inthe extra vascular tissue within a distance of 70 μm from the studiedvessel.

All microscopic observations were made using a Leitz Orthoplanintravital microscope with a Leitz SW25 water immersion objective (LeitzWetslar GMBH, Germany). Images were televised and recorded using aPanasonic WV-1550/G (Panasonic, Japan) video camera.

Results

To transmigrate, the cells start rolling, thereafter start to adheremore firmly, and then migrate out in the surrounding tissue. Plateletactivation factor (PAF) was used to induce this inflammatory process andin normal unstimulated vessels the order of these events were R>A>T. Inthe absence of chemoatractant (PAF) SEQ ID NO 8 [IDX9059] were able toreduce rolling by 81.4% and adherence by 41.9% (FIG. 3 a).

After exposure to PAF the order of events (rolling, adhesion, andtransmigration) was reversed, T>A>R, showing that cells started toadhere and transmigrate. In agreement with that the result shows thatwhen PAF was added, transmigrating cells from a basal level of 1.7cells/field (before adding PAF, FIG. 3 a), reached to 20.66 (afteradding PAF, FIG. 3 b). there was a reduction of rolling cells from 22.3cells/field (before adding PAF, FIG. 3 a) to 7.7 (after adding PAF, FIG.3 b), and adhering cells stayed at about 14 cells/field. In thiscondition SEQ ID NO 8 [IDX9059] reduced the rolling, adherence andtransmigration after PAF exposure. The reduction was for rolling cells48.4%, adherent cells by 28.5% and transmigrating cell by 54.4%, showingthat SEQ ID NO 8 has anti-inflammatory properties which could beeffective on different levels of inflammation and acting on differentinflammatory mediators.

Taken together, these results demonstrate the anti-inflammatory effectsof SEQ ID NO 8 [IDX9059]. Surprisingly, SEQ ID NO 8 showed loweringeffects on rolling and adherence values in the absence of chemotacticagent. This is one indication that this sequence has potential to beused in clinical situations where inhibition of PMN infiltration ispreferred.

4. Study of the Inhibitory Effect of Immunomodulatory Oligonucleotideson Cerebral Ischemic Damage in an Experimental Rat Model

The objective of the study was to investigate the inhibitory effect onischemic brain damage by immunomodulatory oligonucleotides in anexperimental rat model of cerebral ischemia. The animal study wasconducted at the Facility for Division of Experimental VascularResearch, Department of Clinical Sciences, Lund University, Lund,Sweden.

Introduction

It has been shown that, tolerance against ischemic injury can be inducedby LPS via TLR4 in various organs such as heart, brain, and kidney(Heemann et al 2000, Rowland et al, 1997; Tasaki et al, 1997). Althoughthe mechanism of protection is not well understood, the paradigm is thata small inflammatory response by LPS-preconditioning mitigates thesubsequent damaging of inflammatory response associated with a morepowerful secondary stimuli. There are both similarities and differencesamong the known TLR signalling pathways and both TLR4 and TLR9 areexpressed by some cells of immune system and central nervous system(McKimmie and Fazakerley 2005; Tang et al, 2007). Accordingly, CpGoligodeoxynucleotides are believed to trigger activation of the TLR9within selected cell populations to promote innate immunity and induceTh1 biased adaptive immunity.

One aim of this study was to investigate whether the specific compoundsidentified by the present inventors also could decrease ischemic injuryin the brain similar to LPS-induced tolerance to ischemic brain injury.

Materials and Methods

Animal material and conditions: The rats used were inbred WistarHannover rats obtained from Harlan Horst, the Netherlands. The weight ofeach rat was approximately 350-400 grams. The rats were maintained instandard open cages of type Macrolon 3. Cages were housed in open racksunder continuous air flow behind plastic curtains. Standard bedding waspurchased from Scanbur—BK, Sollentuna, Sweden. Bedding was changed oncea week. The temperature in the animal rooms was 18° C.-22° C. and wascontrolled via the ambient ventilation system in the laboratory. Thelight cycle was 12-hour dark and 12-hour light (lights on 06.00).

The rats were given normal rat diet purchased from Scanbur—BK,Sollentuna, Sweden. Water bottles were refilled when necessary duringacclimatization and experimentation. Diet and water were administered adlibitum.

The rats had FELASA SPF-status and the housing and changing system wasdesigned to assure that the SPF-status would be preserved during thestudy. Educated personnel handled the rats. Veterinary expertise wasavailable on short notice from the Veterinary Department at LU. Dailyrecords and decisions made concerning animal welfare.

Test Compounds: IDX9010, IDX9054, IDX9059, and IDX9074 (SEQ ID NO 2, and7-9, respectively, in Table 1) were tested for their in vivo efficacy ofreducing brain damage in an experimental rat model of cerebral ischemia.In addition, a mitogen activated protein kinase inhibitor[1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene; C₁₈H₁₆N₆S₂](U0126, Sigma-Aldrich), and PBS (Invitrogen) served as positive andnegative controls, respectively. A combination treatment of IDX9059 andU0126 was also used at a later stage in the study. All IDX substanceswere synthesized by Biomers.net GmbH, Ulm, Germany under non-GMPconditions and provided in PBS solution. The test compounds were storedat −20° C. as stock solutions. With exception of U0126, and thecombination therapy (U0126+IDX9059), all other test substances weregiven in a blinded manner.

Formulation: Prior to the initiation of an experiment the workingconcentration (1 μg/μl) was prepared by further dilution of thecompounds in PBS at room temperature. The concentration was adjusted byaid of UV spectrophotometry (SmartSpec™ 300, Bio-Rad, Hercules, USA) to95% accuracy.

Animal experiments and dosage: The rats were anesthetized by isofluran(4.5%) mixed with NO₂:O₂ (70%: 30%). Laser Doppler was used to monitorthe cortical blood flow of middle cerebral artery (MCA). A filament wasintroduced into the internal carotid artery, forwarded until it occludedthe blood flow in the right MCA; a more than 80% reduction in the LaserDoppler signal (cortical blood flow) confirmed the occlusion.

The blood pressure, blood gases and blood glucose were controlled duringthe operation. After 90 minutes of occlusion the filament was removedand the circulation of blood continued in the MCA and the cortical bloodflow restituted (reperfusion). Only the animals that had more than 80%decrease in blood flow following the occlusion and high recirculation ofcortical blood flow were included in the study.

The body temperature and the neurological behaviour (score) werecontrolled under the operation and at 0, 1, 2, 24, 48 hours afterrecirculation. A 100 μl of the test compounds together with 200 μl ofPBS were injected intraperitoneal to the rats on 0 and 24 hours afterrecirculation. The animals were sacrificed 48 hours after the end of theoperation, and the brain quickly removed and chilled in bicarbonatebuffer solution. Two mm thick coronal slices (6 slices) of the brainwere prepared and stained with 1% 2,3,5-triphenyltetrazolium chloride(TTC; Sigma Aldrich) dissolved in saline solution. The infarct volumewas calculated by numerical integration of the ischemic area of eachslice using the trapezium rule and was expressed as percentage of totalbrain volume in the slices using the software program Brain DamageCalculator 1.1 (MB Teknikkonsult, Lund, Sweden).

Statistics: Statistical analyses were performed using Prism (Graphpadversion 4.03, San Diego, Calif., USA). Nonparametric, Mann-Whitneyt-test was used to calculate statistical significance. P value of lessthan 0.05 was considered as statistically significant (*).

Results

The induction of tolerance to ischemic cell death by specificoligonucleotides was investigated in an in vivo model of cerebralischemia in Wistar Hannover rat.

In total there were 7 different groups in the study. A total of about 60rats were used in the study with 30% mortality due to the operation.Data were collected throughout the experiments, both in conjunction withthe operation and in the follow up period.

Following TTC staining the intact brain tissue was bright red whileregions of damage were pale—white (FIG. 4 a). The brains were sectionedand each section was photographed and analyzed with an image program(see Methods). The figures describe various aspects of the cerebralinfarct; total infarct area (FIG. 4 b), the penumbral area or selectivenerve necrosis (SNN) (FIG. 4 c), and the ischemic core (the white areaas shown in FIG. 4 a, interpreted as the core) (FIG. 4 d).

The study was performed in a blinded manner for IDX9010 (substance A),IDX9074 (substance B), IDX9054 (substance C), IDX9059 (substance D), andPBS (substance E), but not for U0126 or the combination treatment. Ascan be seen in FIGS. 4 b and 4 d, there was marked reduction (35-40%) ininfarct volume by substance D (IDX9059) and the control substance U0126.There was no significant difference in reduction of SNN with differentsubstances (FIG. 4 c).

After unblinding the study, the last group introduced in the study wasthe combination of IDX9059 and U0126. The reduction was not as expectedadditive but less than that seen by each of the substances alone.

The physiological parameters during the experiment and the result of aneurological evaluation were recorded. There was no difference in thephysiological parameters between the groups (data not shown). However,the neurology evaluation correlated well with improvements by SEQ ID NO8 [IDX9059].

The results show that treatment with the SEQ ID NO 8 [IDX9059] afterinduction of the stroke can serve as post-conditioning and protect thebrain against ischemic injury.

It has previously been shown that the MEK1/2 inhibitor (U0126) canreduce the infarct size by 30-40% (Henriksson M, 2007, Wang Z Q, 2004).In the present study this substance served as a positive control andwhen administered i.p., reduced the infarct volume by 33%. There wasalso a good effect upon the neurology evaluation with improvement byU0126 at 48 hours; thus, confirms that this experiment conducted inaccordance with other investigators (Henriksson et al., 2007).

Recently, Stevens et al. (2008) showed that preconditioning withCpG-containing oligonucleotides may induce neuroprotection againstischemic injury and this tolerance is TNF-alpha dependent. Among the 4inventive compounds (IDX9010, IDX9074, IDX9054, and IDX9059) used inthis study, IDX9059 and IDX9074 showed a better neuroprotective effect.However, four oligonucleotides have previously been used in Wistar ratssplenocytes studies to evaluate the expression of TNF alpha usingquantitative PCR. The results showed that IDX9054, and IDX9010 were ableto induce expression of TNF alpha mRNA, whereas IDX9059, and IDX9074failed to induce TNF alpha. Thus may suggest that there are otherfactors required, in addition to TNF alpha, that collectively provideneuroprotection. In addition, in the study performed by Steven et al.(2008), the protective effect of CpG-containing oligonucleotides wasonly seen when it administered prophylacticalyl-14 days before inductionof stroke. In the present study, the inventive drugs were giventherapeutically 90 min after induction of the stroke, underlining thepotency of the SEQ ID NO 8 [IDX9059].

After unblinding of the present study, SEQ ID NO 8 [IDX9059] proved tobe most effective at reducing ischemic damage in the brain. As thepositive control substance U0126 equally had a pronounced effect atreducing ischemic damage, a combination treatment of U0126 and SEQ ID NO8 [IDX9059] was also evaluated. The reduction of ischemic injuryobtained by the combination treatment was not as expected and the reasonfor this is unknown. However, it could be that the two substancesantagonized each other's effect.

Thus, this in vivo study demonstrated the neuroprotective effect of theinventive compounds in treatment of stroke in a rat model of cerebralischemia.

5. Study of the Inhibitory Effect of Immunomodulatory Oligonucleotide onMyocardial Ischemic Damage in a Model of Isolated-Perfused Rat Heart

The objective of this study was to investigate the possible inhibitoryeffect of immunomodulatory oligonucleotides on ischemic heart damage ina model of isolated, perfused rat hearts. The animal study was conductedat the animal department, at Ullevål University Hospital, Oslo, Norway.

Introduction

The ability to reduce myocardial infarct size by interrupting myocardialreperfusion with short-lived episodes of coronary re-occlusion hasobtained interest in the reperfusion phase as a target forcardioprotection, a phenomenon termed ischemic post-conditioning (IPost). The mechanism of ischemic post-conditioning—induced protection isnot fully understood, but the procedure has been shown to target theimportant mediators of lethal reperfusion injury by reducing oxidativestress, decreasing intracellular Ca2+ overload, improving endothelialfunction, and attenuating apoptotic cardiomyocyte death (Reviewed inYellon).

Ischemic preconditioning (IPC) intervention is also used to reducemyocardial infarct size in the experimental setting by subjecting theheart to one or more episodes of non-lethal myocardial ischemia andreperfusion prior to the sustained coronary artery occlusion. Suchpreconditioning would be beneficial as prophylactic treatment beforecardiac bypass surgery (reviewed in Yellon). Both post- andpre-conditioning activates similar group of downstream signalingcascades in RISK pathway (reperfusion injury salvage kinase pathway)that mediate ischemic protection in the heart or brain (Pignataro,2008). However, both IPC and IPost need an invasive treatment beingapplied directly to the myocardium in order to achieve cardioprotection,which in some clinical settings can be impractical and harmful.

An alternative more amenable strategy is to apply the cardioprotectivestimulus to an organ or tissue remote from the heart, an approachencapsulated by the phenomenon of remote ischemic preconditioning(RIPC). The actual mechanism through which an episode of brief ischemiaand reperfusion in an organ or tissue exerts protection against asubsequent sustained insult of ischemia-reperfusion injury in a remoteorgan or tissue is currently unclear.

The oligodeoxynucleotides trigger activation of Toll-like receptor 9(TLR9) within selected cell populations to promote innate immunity andinduce Th1 biased adaptive immunity. This property of theoligonucleotides modulate inflammation and may provide protectionagainst infectious and non-infectious diseases. Activation of TLR9 byoligonucleotides also targets a series of protein kinases in the RISKpathway (Sun-Hey Lee 2007) similar to IPost and IPC. In previous example(example 4), the inventors showed that oligonucletide IDX9059 is able toreduce ischemia-reperfusion injury in the brain. Therefore one aim ofthis study was to investigate whether the oligonucleotides which wasused to reduce cerebral injury in the previous study also could exertcardioprotective effects during myocardial ischemia-reperfusion.

Materials and Methods

Animal material and conditions: Male Wistar Hannover rats obtained fromScanbur AS, Nittedal, Norway were used in this experiment. The rats werekept in the central animal stable of Ullevål University Hospital.Properly authorized and educated personnel handled the rats. Dailyrecords were made concerning animal welfare. The weight of each rat wasapproximately 250-350 grams. The rats were randomized into twoexperimental groups (test and control, n=8 in each group).

The rats were maintained in standard open cages with standard bedding.The temperature in the animal rooms was 18° C.-22° C. and was controlledvia the ambient ventilation system in the laboratory (humidity 55-60%).The light cycle was 12-hour dark and 12-hour light (lights on 06.00).

A complete, pellet diet RM3 (Scanbur BK AS, Nittedal, Norway) wassupplied ad libitum, and the rats had free access to fresh drinkingwater bottles.

Test Compounds: The DNA based oligonucleotide IDX9059 (SEQ ID NO 8,Table 1) was synthesized by Biomers.net GmbH, Ulm, Germany (Appendix 1)under non-GMP conditions. The compound was provided in PBS solution andstored at −20° C. as stock solution upon arrival.

Formulation: Prior to the initiation of an experiment the workingconcentration (1 μg/μl) was prepared by further dilution of the stocksolution with PBS (Fluka, Sigma) at room temperature, according to InDexSOPB015. The concentration was adjusted by aid of UV spectrophotometry(SmartSpec™ 300, Bio-Rad, Hercules, USA) to 95% accuracy. The testcompound was kept at 4° C. until use.

Animal experiments and dosage: Test drug (1 μg/μl) or placebo (PBS,vehicle of test drug) was given (100 μl) subcutaneously 24 hrs beforeisolation of the heart. The experiment was performed in a blindedfashion.

Rats were anesthetized by intraperitoneal injection of 5% sodiumpentobarbital (60-80 mg kg-1) and heparinised (500 IU i.p). The heartwas harvested and perfused for 20 minutes for stabilization at constantpressure of 70 mmHg (modified Langendorff mode at 37° C.) using KrebsHenseleit buffer as perfusate (mmol/L: NaCl 118.5; NaHCO₃ 25; KCl 4.7;KH₂PO₄ 1.2; MgSO₄/7H₂O 1.2; glucose/1H₂O 11.1; CaCl₂ 1.8). The bufferwas gassed with 95% O₂ and 5% CO₂ to provide oxygen and maintain at aphysiological pH. The heart temperature was kept constant during theexperiment by the surrounding glass tube perfused with water from aheating chamber. A fluid-filled latex balloon (Hugo SachsElektronik-Harvard Apparatus GmbH, Hugstetten, Germany) was insertedinto the left ventricle to measure ventricular pressures by a Powerlabsystem (AD Instruments Pty Ltd, Castle Hill, NSW 2154, Australia). Leftventricular end-diastolic pressure (LVEDP) was set to 5-10 mmHg, andchanges in LVEDP were measured. Left ventricular developed pressure(LVDevP=left ventricular systolic pressure (LVSP)−LVEDP) and maximum andminimum of left ventricular pressure development (LVdp/dtmax andLVdp/dtmin) were calculated. Coronary flow (CF) was measured by timedcollections of the coronary effluent. Arrhythmias were counted as an allor nothing response (asystolia or ventricular fibrillation) during thefirst 30 minutes of reperfusion and evaluated from pressure curves asheart rate (HR). Myocardial temperature was measured by inserting atemperature probe in the right ventricle.

Hearts with LVSP≦100 mmHg, CF≦8 or ≧20 ml min-1, HR≦220 beats per minutebefore ischemia or irreversible arrhythmias for more than 30 minutesduring reperfusion were excluded from the study.

At the end of reperfusion the hearts was cut in four slices of one mmand three slices of two mm (hearts fixed in acrylic rat brain matrix byAgnThor's AB, Lidingö, Sweden). The two mm slices were frozen clamped inliquid nitrogen and stored for possible later analyses (Western blot,real time PCR).

The four ventricular one mm slices (5-8 mm from apex) were incubated at37° C. in 1% triphenyltetrazoliumchloride (TTC) in PBS for 15 minutes.After incubation the slices were gently pressed between two glass platesand photographed (Nikon, Colorfix5400). The infarct area was measured aspercentage of total area and calculated with Adobe Photoshop andScionImage (Infarct Area Calculation Macro file, Copyright © 1998 RobBell, Hatter Institute, UCL, UK).

Extension of necrosis (infarct size) as primary endpoint and heartfunctions (LVEDP, LVSP, LVDevP, LVdp/dtmax, LVdp/dtmin, HR, arrhytmias,CF) as secondary endpoints were evaluated.

Statistics: For infarct size: Students' t-test, for continuous dataanalysis of variance repeated measurements.

Results

The induction of tolerance to ischemic cell death by IDX9059 wasinvestigated in an ex vivo model of isolated rat heart. The hearts wereexcised and perfused in a Longendorff mode and were subjected to anepisode of ischemia and reperfusion as described in Material andMethods. Following TTC staining the hearts were sectioned and eachsection was photographed and analyzed with an image program (seeMethods). The infarct injury of the left ventricle showed 35% reductionin the rats treated with IDX9059 compared to rats treated with PBS(vehicle control) (FIG. 5). Various aspects of the heart function duringreperfusion were also investigated, however no significant difference infunctional data has been observed in IDX9059 treated animals versuscontrol (data not shown).

The results clearly show that subcutaneous pre-treatment of rats withIDX9059 is capable of significantly reducing the extent of globalischemic damage, here by 35%.

The inventors have previously observed that the oligonucleotide IDX9059reduced the ischemic core in a rat model of cerebral ischemia. In thepresent study pre-treatment of rats with IDX9059 in a model ofisolated/perfused rat hearts was found to increase tolerance and reducethe extent of ischemic injury.

In the isolated heart model many of the factors that may be beneficialare not present, such as the effect via leukocytes. Consequently it isexpected that in vivo studies may result in a more pronounced effect.

In the isolated heart the observed protection of IDX9059 may be due tomechanisms initiated in the heart itself. It is reasonable to believethat this is due to molecular changes in the cardiomyocytes via apreconditioning-like stimulation in terms of signalling and effects,following pre-treatment with IDX9059. This could be mediated bydifferent components such as heat shock proteins, nuclear factor kappaB, protein kinases (Hausenloy et al., 2005, and 2007, Valen G 2003, and2005).

The experiment showed that treatment with IDX9059 reduces PMN migrationin different examples above, and indicates that the tested compound alsoexerts a protective effect through other mechanisms such aspreconditioning.

6. Inhibition of Neutrophil Accumulation in Mesenteric Ischemia byImmunomodulatory Oligonucleotides

Summary

Intestinal ischemia was induced in mice by occlusion of the mesentericartery. After reperfusion, damage to small and large intestine wasevaluated, as well as to the lung.

The anti-inflammatory effect of IDX0150 (SEQ ID NO 13, table 1) wasinvestigated by histology, MPO-assay, tetrasolium reduction and smallintestine fluid accumulation.

The study showed that IDX0150 demonstrated significant amelioratingeffect compared to control animals given vehicle only when administered20 minutes before induction of ischemia.

The results indicate that the IDX0150 could be valuable for reduction ofreperfusion damage in ischemia.

Materials and Methods

Test Compounds

All compounds were stored at −20° C. as stock solutions and prepared 2-3days prior to the initiation of an experiment.

Formulation

The oligonucleotides were further diluted in PBS (Fluka BiochemikaUltra, Sigma Aldrich, St. Louis, USA) at room temperature. Theconcentration was adjusted by aid of UV spectrophotometry (SmartSpec™3000, BIO-RAD, Hercules, USA) to 95% accuracy, until the desiredconcentration was reached (InDex SOP B015).

Animal Experiments

Animal Department

The MTC animal department is monitored and supervised by the KarolinskaInstitute Veterinary Department. The animal department has set routinesto maintain a high quality animal facility. The animal study was carriedout in a non-GLP accredited academic research laboratory.

Animals

Female BALB/cJ SPF mice, age 10-30 weeks, (originating from The JacksonLaboratory, Bar Harbor, Me., USA were kept at MTC's CFGR department atKarolinska Institutet, Stockholm). The animals were grouped and allowedto acclimatize for at least one week before the start of an experiment.The animals were kept together with sentinels who were tested accordingto FELASA regulations (5) at a minimum twice a year.

Housing

The animals were kept in rooms at 21±3° C., and with a relative humidityof 55±15%. The ventilation system has been designed to give 10 airchanges per hour. The room was illuminated to give a cycle of 12 hourslight and 12 hours darkness. Light was on from 06:00 to 18:00 hours.

The mice were kept in transparent polycarbonate (Macrolone type III,Scanbur AB, Sollentuna, Sweden) cages (floor area: 810 cm²), 8 in eachcage.

Bedding

The bedding in the cages was Scanbur Bedding (Scanbur AB, Sollentuna,Sweden).

Environmental Enrichment

For environmental enrichment, the animals were given a supply ofSizzle-nest or Happi-Mat, (Scanbur AB, Sollentuna, Sweden)

Diet and Drinking Water

A complete, pellet rodent diet, R36 (Lantmännen, Kimstad, Sweden) wassupplied ad libitum. The animals had free access to animal drinkingbottles with domestic quality drinking water.

Animal Identification, Grouping and Treatment

Each cage was identified by a cage card marked with study number, groupnumber, and sex. The animals were individually marked on the tail withtransverse lines corresponding to the animals' number, using apermanent-ink felt pen.

Test Procedure

Induction of Ischemia and Reperfusion

The mouse was anesthetized with Isofluran (Forene®, Abbott ScandinaviaAB, Solna, Sweden) thereafter kept on surgical anesthesia (Univentor 400anesthesia unit, AgnTho's AB, Lidingo, Sweden) and placed on a heatingpad, controlled by a thermocoupled thermometer (Pharmacia AB, Uppsala,Sweden) controlled by a rectal thermistor maintaining a body temperatureof 37° C. With the aid of an operating surgical stereomicroscope (LeitzWild, Wetslar, Germany) a 4-5 cm long incision was made in the abdomenand the cranial (superior) mesenteric artery was localized. A microvessel clamp, Biemer-clip, closing force 0.20-0.25 N, Aesculap-Werke AG,Tuttlingen, Germany) was placed over the artery to totally occlude theblood stream indicated by lack of pulsation and paleness. The abdomenwas closed and a gauze pad soaked with saline (0.9% (w/v⁻¹ NaCl)solution was placed over the abdomen. The blood flow was reinstatedafter 15 minutes, indicated by return of pulse and redness in thevessel. The abdomen was closed with agraffes or surgical sutures. Themouse was given 2 mL sterile saline s.c. to maintain physiologicalconditions. Buprenorphin (Temgesic®, Schering-Plough Corp., New Jersey,USA), 0.05 to 0.1 mg/kg was given for analgesia. After 3 hours, theanimal was anesthetized, and blood was sampled from the eye orbitalplexus. Samples were taken from intestines and liver for histology andother analyses.

Pharmacological Treatment

Subcutaneous (s.c.) injections of 50 μg/100 μL IDX0150, were given inthe animal's neck about 20 minutes before induction of ischemia orimmediately after starting of reperfusion.

Clinical Signs

Each mouse was observed regularly until killed. All signs of illness,health and any behavioral changes were recorded.

Clinical Parameters

The inflammatory effects were graded using an inflammatory scoringsystem, intestinal redness: normal 0, little redness 1, red 2, very red3; intestinal fluid: normal 0, little 1, much 2; animal behavior: alert0, lethargic 2.

Homogenization of Lung and Intestinal Tissue for MPO Measurement

Lungs and small intestine (100-200 μg) were collected from IDX0150 andPBS-treated mice subjected to intestinal ischemia and reperfusioninjury. The tissues were homogenized for 30 seconds on ice usingDisperser T 10 (IKA®-Werke GMBH & Co. KG, Staufen, Germany) in 1 ml RIPAbuffer (Sigma Aldrich, St. Louis, Mo., USA) containing 5 mM EDTA (SigmaAldrich) and protease inhibitor cocktail (Sigma Aldrich). The sampleswere thereafter incubated on ice for 30 min, after which debris wasremoved from the homogenates by two rounds of centrifugation at 10,000×gfor 10 min at 4° C. The supernatants were collected, aliquoted andfrozen at −70° C. for later myeloperoxidase (MPO) measurements. MPO wereanalyzed in the homogenates using a MPO ELISA kit (Hycult biotechnology,Uden, Holland) according to the manufacturer's instructions.

Results

The ischemia induced a profound inflammatory response which was seen aslethargy and pile erection of the fur, upon necropsy the intestines werebloated, containing fluid and were inflamed (FIG. 6 a). In the IDX0150treated animal the intestine was less inflamed (FIG. 6 b). Using ascoring system the inflamed animals had a score of 6.5 which wascontrasted by a lower score in animals receiving i.p. IDX0150 treatment(FIG. 6 c).

Myeloperoxidase (MPO) is a heme protein abundantly expressed inpolymorphonuclear neutrophils and is used as a marker of neutrophilsinfiltration and activation (Lau and Baldus, 2006). Neutrophils are themajor leukocytes infiltrating the ischemic tissue and are an importantcontributor to the induced inflammation.

In this study the effect of the oligonucleotide compound IDX0150 (SEQ IDNO 13, Table 1) on the infiltration of neutrophils into small intestineand lung in a mouse model of intestinal ischemia and reperfusion injurywas investigated by measuring MPO in homogenized tissue. The MPO levelsin both intestinal (FIGS. 6 d, and e) and lung (FIGS. 6 f, and g) tissuewere reduced in mice treated with IDX0150 compared with PBS treatedcontrols. This decrease in MPO was seen if the compound was administeredbefore induction of ischemia (FIGS. 6 d, and f) as well as afterstarting the reperfusion (FIG. 6 e, and g). The observed reduction inMPO indicates that there is a reduced infiltration of neutrophils intothe ischemic tissue in response to the oligonucleotide compounds of theinvention.

These results were also reflected in the clinical results with areduction in inflammatory score.

7. The Effect of Immunomodulatory Oligonucleotides on the Expression ofChemokine Receptors on Polymorphonuclear Cells

Introduction

IL-8 is a potent proinflammatory cytokine that has a key role in therecruitment and activation of neutrophils during inflammation. IL-8reacts with neutrophils via two distinct types of receptors (CXCR1 andCXCR2). Chemotaxis of neutrophils to IL-8 is mediated predominantly byCXCR1 (78%) and the rest by CXCR2. Another potent chemoattractant forneutrophils is Leukotriene B₄ (LTB₄), which binds with high affinity tothe receptor BLT1 expressed on the surface of neutrophils. The aim ofthis study was to investigate whether the inventive compounds are ableto decrease the expression of CXCR1/2 and BLT1 and thereby reducing theinfiltration of PMN.

Materials and Methods

Test Compounds

IDX9005, IDX9010, IDX9022, IDX9030, IDX9031, IDX9045, IDX9052, IDX9054,IDX9059, IDX9074, IDX9092, IDX9095, IDX9096 and IDX0150 (SEQ ID NO 1 to14, Table 1) were investigated for their effect on CXCR1 and CXCR2surface expression in healthy polymorphonuclear cells (PMN). Theinventive compounds IDX9022, IDX9052, IDX9054 and IDX9059 (SEQ ID NO 3,14, 7 and 8, Table 1) were further investigated for their effect on BLT1surface expression in PMN from healthy volunteers. All oligonucleotideswere synthesized by Biomers.net (Ulm, Germany), except IDX0150 which wasordered from Avecia (Manchester, United Kingdom).

Formulation

The oligonucleotides were adjusted with phosphate buffered saline (PBS,Invitrogen, Carlsbad, Calif.) to reach a stock concentration of 500 μMby aid of UV spectrophotometry (SmartSpec® 3000, BIO-RAD, Hercules, USA)and stored at −20° C. until used.

Cell Preparation and In Vitro Stimulation with Oligonucleotides

Whole blood from healthy blood donors were used for preparation of PMN.PMN were isolated by density centrifugation using Polymorphprep™(Axis-Schield, Oslo, Norway). The cells were then further washed in PBS,and the viability and the cell number were determined by counting thecells in Trypan blue (Sigma Aldrich, Stockholm, Sweden). Thereafter, thecells were re-suspended in complete cell medium consisting of RPMI 1640(Sigma Aldrich) supplemented with 10% heat inactivated fetal calf serum(FCS, Invitrogen), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mLstreptomycin, 10 mM HEPES (Sigma Aldrich) and 5 μg/mL gentamicin(Invitrogen). The PMN were cultured in 96-well culture tissue plates(Becton Dickinson, Franklin Lakes, N.J.) at a concentration of 2×10⁶cells/mL with 0.5, 10 μM or 25 μM of the inventive compounds or withmedium alone as a negative control in a total volume of 200 μl/well. Thecells were incubated for 3 h, if not stated otherwise, at 37° C. in ahumidified cell culture incubator (Thermo Scientific, Waltham, Mass.)with 5% CO₂, after which the cells were analyzed for CXCR1, CXCR2 andBLT1 expression using flow cytometry as described below.

Kinetic Evaluation of the Effect of Oligonucleotides on CXCR1 SurfaceExpression

Human PMN from healthy blood donor were stimulated with 10 μM of IDX9059(SEQ ID NO 8, Table 1) or with medium alone for various time points (15min, 30 min, 1 h, 2 h, and 3 h). Cells were subsequently harvested andfixated at each time point in 2% paraformaldehyde, after which they wereanalyzed for CXCR1 expression by flow cytometry as described below.

Flow Cytometry

Cells incubated with the inventive compounds were harvested, washed inPBS and resuspended in PBS supplemented with 2% FCS. The cells werestained for the granulocyte marker CD66b together with CXCR1 or CXCR2 orBLT1 using fluorochrome labeled mouse monoclonal antibodies (BectonDickinson, San Jose, Calif., USA) for 30 min at 4° C. The antibodiesused were compared with isotype matched controls (Becton Dickinson).After washing in PBS, the cells were analyzed by a FACSarray flowcytometer (Becton Dickinson) and the data were analyzed using theFACSarray software system (Becton Dickinson). A minimum of 15 000 gatedPMN were analyzed per sample.

Chloroquine Assay

PMN isolated from healthy blood donors were pretreated with 0.5, 5 and10 μg/ml of Chloroquine (Sigma Aldrich) for 30 min at 37° C. beforestimulated with 10 μM of test substances for an additional 3 h. Surfaceexpression of CXCR1 and CXCR2 were then analyzed by flow cytometry asdescribed above.

Chemotaxis Assay

Chemotaxis of PMN was investigated using the QCM™ 3 μm 24-wellcolorimetric chemotaxis assay (Millipore, Temecula, Calif.) according tothe manufacturer's instructions. Briefly, PMN were prepared from wholeblood from healthy donors and resuspended in complete cell medium asdescribed above. PMN were pre-incubated in 48-well plates (BectonDickinson) at a concentration of 1×10⁶ cells/mL, using 250 μl cellsuspension per well, with 0.5, 10 or 25 μM of the inventive compoundsfor 1 h at 37° C. in a humidified cell culture incubator (ThermoScientific) with 5% CO₂. The cells were then washed in complete cellmedium and the cells were transferred to the top inserts of a 24-wellcell migration plate assembly having a pore size of 3 μm (Millipore). Tothe lower chambers, complete cell medium containing 100 ng/ml ofrecombinant human IL-8 (Invitrogen) or 500 nM LTB₄ (Sigma Aldrich) wereadded. In one experiment testing IDX9045 the cells were not washedbefore added to the top inserts of the chemotaxis plate and in this caseIL-8 were added together with IDX9045, at the same concentration as usedfor the top insert, to the lower chamber, this to eliminate the risk ofcreating a gradient of the compound. The cells were then allowed tomigrate through the filter towards the chemoattractant for 3 h at 37° C.in a humidified cell culture incubator (Thermo Scientific) with 5% CO₂.Thereafter, the cells from the lower chamber, i.e. migrated cells, weredetected by incubation with the cell viability stain WST-1 for 1 hfollowed by quantification by measuring the absorbance at 450 nm using amicroplate reader (Tecan, Männedorf, Switzerland).

Result and Discussion

PMN are the major leukocytes that are attracted to the ischemic tissue.Here, neutrophils add to the tissue damage by releasing free radicals,proteolytic enzymes such as myeloperoxidase and by stimulating cytokinerelease from local cells leading to increased inflammation. IL-8, bybinding to its receptors CXCR1 and CXCR2, and LTB₄, by binding to itsreceptor BLT1 on the PMN cell surface, play a key role in therecruitment of neutrophils to the inflammation site (Kobayashi, 2008;Tager and Luster, 2003).

In this study the effect of oligonucleotide compounds on the surfaceexpression of CXCR1, CXCR2 and BLT1 on PMN isolated from healthy blooddonors were investigated. After 3 h of stimulation with test substancesseveral of the inventive compounds decreased the mean fluorescenceintensity (MFI) of CXCR1, i.e. the amount of CXCR1 expressed per cell(FIG. 7 a). The largest reduction in CXCR1 expression was induced byIDX9052, IDX9054, IDX9005, IDX9030, IDX9059, IDX9022, and IDX9045 (SEQID NO 14, 7, 1, 4, 8, 3, and 6, Table 1), by which the MFI was decreasedby 68%, 62%, 61%, 60%, 53%, 52% and 30%, respectively when used at 25μM. In addition, there was a small decline in the percentage of CXCR1+cells in PMN stimulated with IDX9005, IDX9022, IDX9030, IDX9052, IDX9054and IDX9059 (SEQ ID NO 1, 3, 4, 14, 7 and 8, Table 1) however, thereduction was not as substantial as for the MFI (FIG. 7 b). CXCR2 wasalso down-regulated in response to the inventive oligonucleotidecompounds (FIG. 7 c), especially when PMN were stimulated with IDX9052and IDX9054 (SEQ ID NO 14 and 7, Table 1), which induced a decrease inthe MFI of CXCR2 by 50 and 36%, respectively. IDX9052 and IDX9054 alsoinduced a small decrease in the percentage of CXCR2+ PMN (FIG. 7 d). Thereduction in CXCR2 expression was dose-dependent with the biggest effectseen at 25 μM of oligonucleotide compound and with less effect seen at10 and 0.5 μM (FIG. 7 e). In addition, in a separate experiment usingPMN from healthy blood donors, IDX9074 (SEQ ID NO 9, Table 1) were ableto down-regulate the expression of CXCR1 (FIG. 7 f).

To investigate the kinetics for CXCR1 down-regulation, human PMN werestimulated with 10 μM of IDX9059 (SEQ ID NO 8, Table 1) for 15 min, 30min, 1 h, 2 h and 3 h after which CXCR1 expression was investigated withflow cytometry. Already after 15 min, a small decrease of CXCR1 surfaceexpression could be observed and after 2 h the down-regulation hadreached maximum level with no further decrease seen after 3 h (FIG. 7g).

The inventive compounds IDX9052, IDX9054, IDX9059, IDX9005 and IDX9045(SEQ ID NO 14, 7, 8, 1, and 6) were all very efficient in reducingCXCR1/2 expression and have in common that they all contain a G-flank intheir 5′- and/or 3′-end, suggesting that this structural motif, togetherwith the sequence of the oligonucleotide, could be associated with amore efficient CXCR1/2 reducing capacity by the oligonucleotides. Tofurther support this, when PMN from healthy blood donors were incubatedwith the control oligonucleotides IDX0480 (T*G*C*TGCTTCTGCCATGCTG*C*T*T)and IDX9134 (G*A*T*GCTCTG*G*G*G), which have the same sequences asIDX9022 and IDX9059, respectively, but without CpG motifs, IDX0480 wasunable to reduce the surface expression of CXCR1 as seen for IDX9022,while IDX9134 was equally potent as IDX9059 in reducing CXCR1 surfaceexpression (FIG. 7 h). Without wishing to be bound by any theory, theinventors contemplate that these results indicate that IDX9022, whichdoes not have a G-flank, mediates its CXCR1 and CXCR2 reducing effectmainly through its CpG motifs, while IDX9059 containing a G-flank hasCpG-independent effects.

To investigate the role of TLR9 in the reduction of CXCR surfaceexpression induced by the test substances, PMN were pretreated withChloroquine before stimulated with test substances. Chloroquine is a4-aminoquinoline drug, which blocks endosomal fusion and acidificationand prevents TLR9 activation and downstream metabolic signalingpathways. Chloroquine could dose-dependently inhibit the reduction inCXCR1 surface expression induced by IDX9059 and IDX9022 (SEQ ID NO 8 and3, Table 1) (FIG. 7 i). 10 μg/ml of Chloroquine almost completelyblocked the decrease in CXCR1 expression induced by these two compounds(FIG. 7 i). However, chloroquine could not block the reduction in CXCR1surface expression induced by IDX9054 or IDX9052 (SEQ ID NO 7 and 14,Table 1) (FIG. 7 i). These results indicate that some of the inventivecompounds mediates their CXCR reducing effect through endosomal TLR9activation (i.e. IDX9022 and IDX9059), while others do not (i.e. IDX9052and IDX9054).

A dose-dependent reduction of BLT1, the receptor for the chemoattractantLTB₄, could also be demonstrated after incubation with differentconcentrations (0.5. 10 and 25 μM) of the inventive compounds (FIG. 7j). An 87%, 80%, 64% and 57% reduction in the MFI was seen with theinventive compounds IDX9052, IDX9054, IDX9059 and IDX9022 respectively(SEQ ID NO 14, 7, 8 and 3, Table 1) when used at 25 μM (FIG. 7 j). Inaddition, IDX9052 and IDX9054 significantly reduced the percentage ofBLT1 positive PMN (FIG. 7 k). As shown in FIG. 7 j the inventivecompounds containing a G-flank in their 5′- and/or 3′-end, i.e. IDX9052,IDX9054 and IDX9059 (SEQ ID NO 14, 7 and 8, Table 1) were more efficientin reducing BLT1 surface expression, compared to IDX9022 which does nothave a G-flank.

To investigate if the reduction in CXCR1, CXCR2 and BLT1 surfaceexpression induced by the inventive compounds also leads to decreasedmigration of PMN towards the CXCR1 and CXCR2 ligand (IL-8) or the BLT1ligand (LTB₄), PMN from healthy blood donors were pre-incubated for 1 hwith the inventive oligonucleotide compounds. The cells were thenallowed to migrate towards IL-8 or LTB₄ in a chemotaxis assay for 3 h. Adose dependent reduction in the number of migrated PMN were seen afterpre-incubation with both IDX9022, IDX9052, IDX9054 and IDX9059 (SEQ IDNO 3, 14, 7 and 8, Table 1), with the most efficient compounds beingIDX9052, IDX9054 and IDX9059, which at 25 μM completely or almostcompletely blocked the migration of PMN towards both IL-8 (FIG. 7 l) andLTB₄ (FIG. 7 n). In addition, In a separate experiment using PMN fromtwo healthy blood donors, IDX9045 (SEQ ID NO 6, Table 1)dose-dependently reduced IL-8 induced migration of PMN (FIG. 7 m).

The surface expression of CXCR1 and 2 correlated with the number ofmigrated PMN in the IL-8 induced chemotaxis, as did the surfaceexpression of BLT1 and the number of migrated PMN in the LTB₄ inducedchemotaxis (FIG. 7 o-q). This indicates that the reduction in PMNmigration is due to a lower surface expression of the receptors.

The inventors demonstrated a rapid reduction in CXCR1, CXCR2 and BLT1expression on PMN incubated with oligonucleotide compounds, that forCXCR1 was shown to start already after 15 min of stimulation. This isvery important since neutrophils are terminally differentiated cells andtherefore short lived, why a quickly induced immunomodulatory effect aredesired. Hayashi et al, in 2003, demonstrated a down-regulation of CXCR1expression on PMN by CpG DNA only in cells pretreated with GM-CSF(Hayashi et al., 2003), whereas in this study, the inventiveoligonucleotide substances reduced surface expression of CXCR1 and 2without cytokine pretreatment, underlining their potent immunomodulatoryproperties.

The inventors not only demonstrate a reduction in the surface expressionof CXCR1, 2 and BLT1 but in addition demonstrate a reducedresponsiveness of the cells to the chemoattractants IL-8 and LTB₄resulting in less migration. There is reason to predict that theseresults also reflect an in vivo scenario, consequently leading to lesschemotaxis of PMN into inflamed tissues.

A decreased surface expression of CXCR1, CXCR2 and BLT1 as well asreduced responsiveness of the cells to the chemoattractants IL-8 andLTB₄ was observed in healthy PMN treated with the inventive compounds.These properties of the oligonucleotide compounds clearly show thepotency of the inventive compounds to influence the properties andbehavior of PMNs.

8. The Effect of Immunomodulatory Oligonucleotides on the Expression ofChemokine Receptors on Polymorphonuclear Cells from Asthmatic and MSPatients

Introduction

PMN is involved in the pathogenesis of not only ischemia, but also ofmany other inflammatory disorders, and blocking PMN functions wouldtherefore be beneficial in many inflammatory diseases. Cellularinflammation of the airways with PMN, i.e. eosinophils and neutrophils,is a characteristic feature of asthma. PMN has also been described to beinvolved in the pathogenesis of Multiple Sclerosis (MS). The aim of thisstudy was to investigate whether the inventive compounds are able todecrease the expression of CXCR1/2 and BLT1 not only in PMN from healthyindividuals, but also in PMN from patients with an inflammatorycondition, in this case exemplified by asthma and MS.

Materials and Methods

Test Compounds

IDX9022, IDX9052, IDX9054 and IDX9059 (SEQ ID NO 3, 14, 7 and 8,Table 1) were investigated for their effect on CXCR1, CXCR2 and BLT1surface expression in PMN from asthmatic and MS patients. Alloligonucleotides were synthesized by Biomers.net (Ulm, Germany).

Formulation

The oligonucleotides were adjusted with phosphate buffered saline (PBS,Invitrogen, Carlsbad, Calif.) to reach a stock concentration of 500 μMby aid of UV spectrophotometry (SmartSpec® 3000, BIO-RAD, Hercules, USA)and stored at −20° C. until used.

Cell Preparation and In Vitro Stimulation with Oligonucleotides

Whole blood from asthmatic and MS patients were used for preparation ofPMN. PMN were isolated, counted and resuspended in complete cell mediumas described in materials and methods under example 7. The PMN werecultured in 96-well culture tissue plates (Becton Dickinson, FranklinLakes, N.J.) at a concentration of 2×10⁶ cells/mL with 0.5, 10 μM or 25μM of oligonucleotides or with medium alone as a negative control in atotal volume of 200 μl/well. The cells were incubated for 3 h at 37° C.in a humidified cell culture incubator (Thermo Scientific, Waltham,Mass.) with 5% CO₂, after which the cells were analyzed for CXCR1, CXCR2and BLT1 expression using flow cytometry.

Flow Cytometry

Cells incubated with oligonucleotides were harvested, washed in PBS andresuspended in PBS supplemented with 2% FCS. The cells were stained forthe granulocyte marker CD66b together with CXCR1 or CXCR2 or BLT1 asdescribed in materials and methods under example 7. The cells were thenanalyzed by a FACSarray flow cytometer (Becton Dickinson) and the datawere analyzed using the FACSarray software system (Becton Dickinson). Aminimum of 15 000 gated PMN were analyzed per sample.

Result and Discussion

PMN are one of the major cells mediating tissue damage during aninflammatory response. PMN migrate from the blood to sites ofinflammation in response to locally produced chemoattractants. Two ofthe major mediators of PMN migration are the CXC chemokine IL-8 and theleukotriene LTB₄. In this study, the inventors set out to investigate ifthe inventive compounds could down-regulate the receptors for IL-8, i.e.CXCR1 and CXCR2, as well as the receptor for LTB₄, i.e. BLT1, on thesurface of PMN derived from patients with an inflammatory disease.

Using blood from MS patients indicated a dose-dependent reduction ofCXCR1, CXCR2 and BLT1 on PMN after stimulations with the inventivecompounds. (FIG. 8 a-f). Similar results were seen when blood from anasthmatic patient was used (FIG. 8 g-i). The reduction was predominantlyseen for the MFI, i.e. the amount of receptors expressed per cell.IDX9052, IDX9054 and IDX9059 (SEQ ID NO 8, 7 and 14, Table 1) induced areduction of the MFI for CXCR1 on PMN from MS patients with 45%, 56% and42% respectively when used at 25 μM (FIG. 8 a). In addition, IDX9052,IDX9054 and IDX9059 reduced the surface expression of CXCR2 with 76%,58% and 39% (FIG. 8 c), and the surface expression of BLT1 with 50%, 47%and 26%, respectively (FIG. 8 e). When stimulating PMN from an asthmaticpatient with the inventive compounds, the reduction in MFI for CXCR1 was56%, 71% and 62% with IDX9052, IDX9054 and IDX9059 (FIG. 8 g),respectively. IDX9052, IDX9054 and IDX9059 also reduced the MFI forCXCR2 on PMN from an asthmatic patient with 69%, 50% and 37% (FIG. 8 h),and the MFI for BLT1 with 85%, 77% and 64% (FIG. 8 i), respectively. Asseen with PMN from healthy donors, the inventive compounds containing aflanking oligo-G sequence, i.e. IDX9052, IDX9054 and IDX9059 (SEQ ID NO14, 7 and 8, Table 1) were the most efficient in reducing CXCR1, CXCR2and BLT1 surface expression also in PMN derived from MS and asthmapatients.

A decreased surface expression of CXCR1, CXCR2 and BLT1 was alsoobserved when PMN derived from patients with an inflammatory disorder,i.e. asthmatic and MS patients, were treated with the inventivecompounds. These properties of the oligonucleotide compounds could beuseful in reducing the infiltration of PMN into inflamed tissues.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionas set forth in the claims appended hereto.

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The invention claimed is:
 1. An isolated oligonucleotide sequenceselected from the group consisting of the oligonucleotide sequences ofSEQ ID NO: 8 (IDX9059); SEQ ID NO: 1 (IDX9005); SEQ ID NO: 9 (IDX9074);SEQ ID NO: 2 (IDX9010); SEQ ID NO: 4 (IDX9030); SEQ ID NO: 10 (IDX9092);SEQ ID NO: 11 (IDX9095); SEQ ID NO: 12 (IDX9096); and SEQ ID NO: 16(IDX9134), wherein the isolated oligonucleotide sequence consists of SEQID NO 8, 1, 9, 2, 4, 10, 11, 12 or 16, and wherein at least onenucleotide has a phosphate backbone modification.
 2. The oligonucleotideaccording to claim 1, wherein the phosphate backbone modification is aphosphorothioate or phosphorodithioate modification.
 3. A pharmaceuticalcomposition comprising an oligonucleotide according to claim 1.